1. Field of the Invention
The present invention relates generally to the diagnosis and treatment of polycystic kidney disease and more specifically to probes and agents useful in diagnosing and treating polycystic kidney disease and related disorders.
2. Background Information
Autosomal dominant polycystic kidney disease (ADPKD), also called adult-onset polycystic kidney disease, is one of the most common hereditary disorders in humans, affecting approximately one individual in a thousand. The prevalence in the United States is greater than 500,000, with 6,000 to 7,000 new cases detected yearly (Striker et al., Am. J. Nephrol. 6:161-164, 1986; Iglesias et al., Am. J. Kid. Dis. 2:630-639, 1983). The disease is considered to be a systemic disorder, characterized by cyst formation in the ductal organs such as kidney, liver, and pancreas, as well as by gastrointestinal, cardiovascular, and musculoskeletal abnormalities, including colonic diverticulitis, berry aneurysms, hernias, and mitral valve prolapse (Gabow et al., Adv. Nephrol. 18:19-32, 1989; Gabow, New Eng. J. Med. 329:332-342, 1993).
The most prevalent and obvious symptom of ADPKD is the formation of kidney cysts, which result in grossly enlarged kidneys and a decrease in renal-concentrating ability. In approximately half of ADPKD patients, the disease progresses to end-stage renal disease, and ADPKD is responsible for 4-8% of the renal dialysis and transplantation cases in the United States and Europe (Proc. Eur. Dialysis and Transplant Assn., Robinson and Hawkins, eds., 17:20, 1981).
Few diagnostics are available for the identification and characterization of mutations of the PKD1 gene, which is located on human chromosome 16. A major factor contributing to the difficulty in identifying and characterizing mutations of the PKD1 gene is that greater than 70% of the length of the PKD1 gene is replicated on chromosome 16 and elsewhere, resulting in at least six PKD1 homologs. Significantly, the PKD1 homologs share a very high sequence identity with the PKD1 gene, including sequences having greater than 95% identity with the PKD1 gene. As such, oligonucleotides that have been examined for use as specific probes, or as primers for amplification, of PKD1 gene sequences have been found to cross-hybridize with the PKD1 homologs, and the inability to identify PKD1 locus specific probes has prevented accurate analysis of PKD1 gene mutations.
The identification and characterization of PKD1 gene mutations have been further hindered, in part, because transcription of the PKD1 gene results in production of a 14 kilobase (kb) mRNA, which is highly GC-rich. In addition, unlike the remainder of the PKD1 gene, which is extremely compact (approximately 13.5 kb mRNA coded within approximately 30 kb genomic DNA), exon 1 is separated from the rest of the gene by an intron of approximately 19 kb. Thus, previous investigators have simply placed the 5′ anchor primer within the first intron and used it as a link to more 3′ sequences. Exon 1 has several other features that have been major obstacles to its amplification, including an extremely high GC content (approximately 85%), and the ability to replicate with high fidelity in PKD1 gene homologs. Furthermore, no effective method for DNA based analysis of PKD1 gene exon 22, which is flanked on both ends by introns that contain lengthy polypyrimidine tracts. Accordingly, very few positions within the replicated segment and flanking exon 22 are suitable for the design of PKD1-specific primers.
A few oligonucleotides useful for examining regions of the human PKD1 gene, have been described. For example, the primer set forth below as SEQ ID NO:11 has been described in U.S. Pat. No. 6,017,717, and the primer set forth as SEQ ID NO:18 has been described by Watnick et al. (Hum. Mol. Genet. 6:1473-1481, 1997). Also, the primers set forth below as SEQ ID NOS:9, 10, 49 to 51, and 61 to 105 have been described by Watnick et al. (Am. J. Hum. Genet. 65:1561-1571, 1999). The primers set forth below as SEQ ID NOS: 9 and 10 and SEQ ID NOS: 11 and 12 also were more recently described by Phakdeekitcharoen et al. (Kidney International 58:1400-1412, 2000). In addition, a primer set forth as SEQ ID NO:13 in U.S. Pat. No. 6,071,717 has a nucleotide sequence that is substantially identical to that set forth below as SEQ ID NO:10, and a primer designated TWR2 by Watnick et al. (Mol. Cell 2:247-251, 1998) has a nucleotide sequence that is substantially identical to that set forth below as SEQ ID NO:12.
Despite the large number of families having diseases associated with PKD1 gene mutations, the potential clinical and scientific impact of mutation studies, and the availability of a genomic structure, the fact that only a relatively small number of PKD1 mutations have been described demonstrates the relative paucity of data due to the complicated genomic structure of the PKD1 gene. Thus, there exists a need for diagnostic methods suitable for examining the PKD1 gene and for identifying disorders related to PKD1 gene mutations. The present invention satisfies this need and provides additional advantages.